The integrity of the genome is of prime importance to a dividing cell. In response to DNA damage, eukaryotic cells rely upon a complex system of controls to delay cell-cycle progression. The normal eukaryotic cell-cycle is divided into 4 phases (sequentially G1, S, G2, M) which correlate with distinct cell morphology and biochemical activity. Cells withdrawn from the cell-cycle are said to be in G0, or non-cycling state. When cells within the cell-cycle are actively replicating, duplication of DNA occurs in the S phase, and active division of the cell occurs in M phase. See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 36, 1997). DNA is organized in the eukaryotic cell into successively higher levels of order that result in the formation of chromosomes. Non-sex chromosomes are normally present in pairs, and during cell division, the DNA of each chromosome replicates resulting in paired chromatids. (See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 5, 1997).
The eukaryotic cell cycle is tightly regulated by intrinsic mechanisms that ensure ordered progression through its various phases and surveillance mechanisms that prevent cycling in the presence of aberrant or incompletely assembled structures. These negative regulatory surveillance mechanisms have been termed checkpoints (Hartwell and Weinert, 1989, “Checkpoints: controls that ensure the order of cell cycle events” Science, 246: 629-634). The mitotic checkpoint prevents cells from undergoing mitosis until all chromosomes have been attached to the mitotic spindle whereas the DNA structure checkpoint, which can be subdivided into the replication and DNA damage checkpoint, result in arrests at various points in the cell cycle in the presence of DNA damage or incompletely replicated DNA (Elledge, 1996, “Cell cycle checkpoints: preventing an identity crisis.” Science, 274: 1664-1672). These arrests are believed to allow time for replication to be completed or DNA repair to take place. Cell cycling in the presence of DNA damage, incompletely replicated DNA or improper mitotic spindle assembly can lead to genomic instability, an early step in tumorigenesis. Defective checkpoint mechanisms, resulting from inactivation of the p53, ATM, and Bub1 checkpoint gene products have been implicated in several human cancers.
Checkpoint delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase (Hartwell and Weinert, 1989, supra.). In many cases the DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases (Elledge, 1997, supra.). In human cells the DNA-damage induced G2 delay is largely dependent on inhibitory phosphorylation of Cdc2 (Blasina et al., 1997, “The role of inhibitory phosphorylation of cdc2 following DNA replication block and radiation induced damage in Human cells.” Mol. Biol. Cell 8: 1013-1023; Jin et al., 1997, “Role of inhibiting cdc2 phosphorylation in radiation-induced G2 arrest in human cells.” J. Cell Biol., 134: 963-970), and is therefore likely to result from a change in the activity of the opposing kinases and phosphatases that act on Cdc2. However, evidence that the activity of these enzymes is substantially altered in response to DNA damage is lacking (Poon et al., 1997, “The role of cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells.” Cancer Res., 57: 5168-5178).
Three distinct Cdc25 proteins are expressed in human cells. Cdc25A is specifically required for the G1-S transition (Hoffmann et al., 1994, “Activation of the phosphatase activity of human CDC25A by a cdk2-cyclin E dependent phosphorylation at the G-1/S transition.” EMBO J., 13: 4302-4310; Jinno et al., 1994, “Cdc25A is a novel phosphatase functioning early in the cell cycle” EMBO J., 13: 1549-1556), whereas Cdc25B and Cdc25C are required for the G2-M transition (Gabrielli et al., 1996, “Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule mucleation in HeLa cells” J. Cell Sci., 109(5): 1081-1093; Galaktionov et al., 1991, “Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins” Cell, 67: 1181-1194; Millar et al., 1991, “p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells” Proc. Natl. Acad. Sci. USA, 88: 10500-10504; Nishijima et al., 1997, J. Cell Biol., 138: 1105-1116). The exact contribution of Cdc25B and Cdc25C to M-phase progression is not known.
Much of our current knowledge about checkpoint control has been obtained from studies using budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast. A number of reviews of our current understanding of cell cycle checkpoint in yeast and higher eukaryotes have recently been published (Hartwell & Kastan, 1994, “Cell cycle control and Cancer” Science 266: 1821-1828; Murray, 1994, “Cell cycle checkpoints” Current Opinions in Cell Biology, 6: 872-876; Elledge, 1996, supra; Kaufmann & Paules, 1996, “DNA damage and cell cycle checkpoints” FASEB J., 10: 238-247). In the fission yeast six gene products, rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+ have been identified as components of both the DNA-damage dependent and DNA-replication dependent checkpoint pathways. In addition cds1+ has been identified as being required for the DNA-replication dependent checkpoint and rad27+/chk1+ has been identified as required for the DNA-damage dependent checkpoint in yeast.
Several of these genes have structural homologues in the budding yeast. Further conservation across eukaryotes has recently been suggested with the cloning of several human homologues of S. pombe checkpoint genes, including two related to S. pombe rad3+: ATM (ataxia telangiectasia mutated) (Savitsky et al., 1995, “A single ataxia telangiectasia gene with a product similar to PI-3 kinase” Science, 268: 1749-1753) and ATR (ataxia telangiectasia and rad3+ related)(Bentley et al., 1996, “The Schizosaccharomyces pombe rad3 checkpoint genes” EMBO J., 15: 6641-6651; Cimprich et al., “cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein” 1996, Proc. Natl. Acad. Sci. USA, 93: 2850-2855); and human homologues of S. pombe rad9+, Hrad9 (Lieberman et al., 1996, “A human homolog of the Schizosaccharomyces pombe rad9+ checkpoint control gene” Proc. Natl. Acad. Sci. USA 93: 13890-13895), Hrad1(Parker et al., 1998, “Identification of a human homologue of the Schizosaccharomyces pombe rad17+ checkpoint gene” J. Biol. Chem. 273:18340-18346; Freire et al., 1998, “Human and mouse homologs of Schizosaccharomyces pombe rad1(+) and Saccharomyces cerevisia RAD17: linkage to checkpoint control and mammalian meiosis” Genes Dev. 12:2560-2573; Udell et al., 1998, “Hrad1 and Mrad1 encode mammalian homologues of the fission yeast rad1(+) cell cycle checkpoint control gene” Nucleic Acids Res. 26:2971-3976), Hrad17 (Parker et al., 1998, supra), Hhus1 (Kostrub et al., 1998, “Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage” EMBO J. 17:2055-2066), Hchk1 (Sanchez et al., 1997, “Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25” Science 277:1497-1501) and Hcds1 (Matusoka et al., 1998, “Linkage of ATM to cell cycle regulation by the Chk2 protein kinase” Science 282(5395):1893-1897; Blasina et al., 1999, “A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase” Curr. Biology 9(1):1-10).
Genetic and biochemical analysis of the checkpoint proteins in yeast and mammalian cells suggests that the checkpoint response is transmitted through a conventional signal transduction pathway. Hrad1, Hrad9, Hrad17, and Hhus1 transmit the signal emanating from damaged or incompletely replicated DNA to the central kinases ATM and ATR, which in turn activate the downstream kinases, Chk1 and Cds1. The DNA structure checkpoint responses ultimately lead to phosphorylation of the mitosis inducing phosphatase Cdc25 by Chk1 or Cds1. This phosphorylation event creates a binding site for 14-3-3 proteins that target Cdc25 for export from the nucleus to the cytoplasm, thus preventing it from removing an inhibitory phosphate from the cyclin dependent kinase, Cdc2. Removal of this inhibitory phosphate is required for passage from G2 to mitosis in every cell cycle. The DNA structure checkpoint responses prevent this from occurring and result in a G2/M arrest.
Whereas the Chk1 protein has been shown to be required for the G2/M DNA damage checkpoint in S. pombe, the replication checkpoint requires the activity of both Cds1 and Chk1. When replication is blocked by treatment with the ribonucleotide reductase inhibitor hydroxyurea (HU), wild type cells arrest prior to mitosis. A cds1chk1 double mutant fails to arrest in the presence of HU while both single mutants arrest normally (Russell, 1998, “Checkpoints on the road to mitosis” Trends in Biochemical Sciences 23(10):399-402). S. pombe Chk1 and Cds1 are both capable of phosphorylating Cdc25 and targeting it for binding by 14-3-3 proteins. Activation of the S. pombe Cds1 protein kinase by HU also results in enhanced binding to and phosphorylation of Wee1, and accumulation of Mik1. These two protein kinases are required for the inhibitory phosphorylation of Cdc2 that prevents cells from entering mitosis suggesting an alternative to Cdc25C phosphorylation for checkpoint mediated cell cycle arrest. Recently, Cds1 has also been shown to be required for a DNA damage checkpoint in S-phase (Rhind and Russell, 1998, “The Schizosaccharomyces pombe S-phase checkpoint differentiates between different types of DNA damage” Genetics 149(4):1729-1737; Lindsay et al., 1998, “S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe” Genes Dev. 12(3):382-395). A human homologue of S. pombe Cds1 that is activated by DNA damage and HU in an ATM-dependent manner and is capable of phosphorylating Cdc25C in vitro was recently identified (Matsuoka et al., 1998, supra; Blasina et al., 1999, supra). The human cDNA encodes a 543 amino acid protein which like its S. pombe homologue, contains a forkhead associated (FHA) domain N-terminal to the kinase domain. FHA domains are found in several other proteins including the S. cerevisiae Cds1 orthologue Rad53. Rad53 contains two FHA domains, one of which is required for interaction with the DNA damage checkpoint protein Rad9 in the presence of DNA damage (Sun et al., 1998, “Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint” Science 281(5374):272-274).
In order to develop new and more effective treatments and therapeutics for the amelioration of the effects of aging or disease such as cancer, it is important to identify and characterize mammalian, and in particular human, checkpoint proteins and to identify mediators of their activity. The present invention teaches the identification and characterization of human and murine nucleic acids encoding human Mus81 (Hmus81) and murine Mus81 (Mmus81) protein with significant homology to the S. pombe Mus81 protein that interacts with the S. pombe Cds1 FHA domain. The S. cerevisiae orthologue is reported to be involved in meiosis and DNA repair.
As described below, a Hmus81 gene acts as a checkpoint/repair gene and is involved with DNA repair. The checkpoint/repair delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase, and Hmus81 appears to act in both aspects, similarly to other known checkpoint/repair genes. In many cases, the DNA-damage response pathways will cause arrest, and the cell will fail to divide. However, a functional DNA repair mechanism will allow the damage to be corrected, and thus allow eventual cell division to occur.
In humans, excision repair is an important defense mechanism against two major carcinogens, sunlight and cigarette smoke. It has been found that individuals defective in excision repair exhibit a high incidence of cancer. (see Sancar, A, 1996, “DNA Excision Repair” Ann. Rev. Biochem. 65:43-81). Other mechanisms also act in a simiar manner to repair DNA, such as mismatch repair which stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences. Inactivation of genes encoding these activities results in a large increase in spontaneous mutability and predisposition to tumor development. (see Modrich & Lahue, 1996, “Mismatch Repair in Replication Fidelity, Genetic Recombination and Cancer Biology” Ann. Rev. Biochem. 65:101-33). The importance of maintaining fidelity in the DNA is amply illustrated by the many mechanisms for repair, and if unrepairable, arrest of cell division. (see Wood, R D, 1996, “DNA Repair in Eukaryotes” Ann. Rev. Biochem. 65:135-67).
Many chemotherapeutic agents are designed to disrupt or otherwise cause damage to the DNA of the targeted malignant cells. Antineoplastic agents such as alkylating agents, antimetabolites, and other chemical analogs and substances typically act by inhibiting nucleotide biosynthesis or protein synthesis, cross-linking DNA, or intercalating with DNA to inhibit replication or gene expression. Bleomycin and etoposide for example, specifically damage DNA and prevent repair.
The inhibition of Hmus81 gene or protein activity amplifies the potency of antineoplastic agents, and enhances the efficacy of their use as chemotherapeutic agents. This enhancement is beneficial in not only more thoroughly affecting the targeted cells, but by allowing for reduced dosages to be used in proportion to the increased efficacy, thus reducing unwanted side effects. Inhibition of Hmus81 or Mmus81 gene activity via anti-sense nucleic acid pharmaceuticals can be effected using the nucleic acids of the invention as the template for constructing the anti-sense nucleic acids. It is preferred to target the amino terminal end of the nucleic acid for anti-sense binding, and thus inhibition, as this reduces translation of the mRNA. Inhibition of Hmus81 protein activity can be effected by the use of altered or fragments of Hmus81 or Mmus81 protein to competitively inhibit the biochemical cascade that results in the repair of damaged DNA, or to cause cell arrest.
Disease can also result from defective DNA repair mechanisms, and include hereditary nonpolyposis colorectal cancer (defect in mismatch repair), Nijmegen breakage syndrome (defect in double strand break repair), Xeroderma pigmentosum, Cockayne syndrome, and Trocothiodystrophy (defect in nuclear excision repair). (see for example Lengauer et al., 1998, “Genetic instabilities in human cancers” Nature 396(6712):643-649; Kanaar et al., 1998, “Molecular mechanisms of DNA double stranded repair” Trends Cell Biol. 8(12):483-489).
It is further envisioned that the transient inhibition of Hmus81 gene or protein activity can be sufficient to effect improved treatment of cell behavior due to aging or disease. For example, the transient inhibition of DNA checkpoint/DNA damage arrest of cell division may allow the combined use of lower doses of chemotherapeutic agents to effect greater damage to targeted cells in the treatment of diseases such as cancer.